1887

Abstract

The human cytomegalovirus (HCMV) UL36–38 immediate early (IE) locus encodes proteins required for virus growth. The UL37 IE promoter drives production of differentially spliced and unspliced RNAs. To study their post-transcriptional processing, we generated target minigenes encoding each UL37 RNA splicing substrate. Target 1 RNA, spanning UL37 exon 1 (x1) donor and 2 (x2) acceptor as well as adjacent intronic sequences, but not the UL38 gene, accurately reproduced UL37 x1/x2 RNA splicing in transfected permissive cells. Surprisingly, deletion of distal intronic sequences nt −82 to −143 from the UL37x2 acceptor resulted in aberrant splicing to an upstream non-consensus exonic donor. Target 1 RNAs carry the UL37x1 polyadenylation (PA) signal and site as well as a downstream SV40 early PA signal. Both the UL37x1 and SV40 PA signals are used in wild-type target 1 RNAs but inhibited in UL37x1 PA signal mutants. Alternative RNA splicing of UL37 exons 2 to 3 or 3A as well as exons 3 to 4, observed in HCMV mature UL37 and UL36 spliced RNAs, is accurately reproduced with target minigene RNAs carrying the corresponding UL37 exonic and intronic sequences. Moreover, alternative splicing using two novel UL37 exon 3 consensus splice donors (di and dii) was found in target and in HCMV-infected cell RNA. These results demonstrate that: (i) target minigene RNAs accurately recapitulate the processing of UL37 IE RNAs in the HCMV-infected cell; (ii) precise UL37x1 donor selection is modulated by 3′-distal UL37 intronic sequences; and (iii) UL37 exon 3 contains multiple alternative consensus splice donors.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.18700-0
2003-01-01
2019-12-06
Loading full text...

Full text loading...

/deliver/fulltext/jgv/84/1/vir840029.html?itemId=/content/journal/jgv/10.1099/vir.0.18700-0&mimeType=html&fmt=ahah

References

  1. Berglund, J. A., Abovich, N. & Rosbash, M. ( 1998; ). A cooperative interaction between U2AF65 and mBBP/SF1 facilitates branchpoint region recognition. Genes & Dev 12, 858–867.[CrossRef]
    [Google Scholar]
  2. Bienroth, S., Wahle, E., Suter-Crazzolara, C. & Keller, W. ( 1991; ). Purification of the cleavage and polyadenylation factor involved in the 3′-processing of messenger RNA precursors. J Biol Chem 266, 19768–19776.
    [Google Scholar]
  3. Borst, E.-M., Hahn, G., Koszinowski, U. H. & Messerle, M. ( 1999; ). Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli: a new approach for construction of HCMV mutants. J Virol 73, 8320–8329.
    [Google Scholar]
  4. Burge, C. B., Tuschl, T. & Sharp, P. A. ( 1998; ). Splicing of precursors to mRNAs by the spliceosomes. In The RNA World, 2nd edn, pp. 525–560. Edited by R. F. Gesteland, T. R. Cech & J. F. Atkins. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  5. Chee, M. S., Bankier, A. T., Beck, S. & 12 other authors ( 1990; ). Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr Top Microbiol Immunol 154, 125–169.
    [Google Scholar]
  6. Colberg-Poley, A. M. ( 1996; ). Functional roles of immediate early proteins encoded by the human cytomegalovirus UL36-38, UL115-119, TRS1/IRS1 and US3 loci. Intervirology 39, 350–360.
    [Google Scholar]
  7. Colberg-Poley, A. M., Voss, S. D., Chowdhury, K. & Gruss, P. ( 1985; ). Structural analysis of murine genes containing homoeo box sequences and their expression in embryonal carcinoma cells. Nature 314, 713–718.[CrossRef]
    [Google Scholar]
  8. Colberg-Poley, A. M., Santomenna, L. D., Harlow, P. P., Benfield, P. A. & Tenney, D. J. ( 1992; ). The HCMV US3 and UL36-38 immediate early proteins regulate gene expression. J Virol 66, 95–105.
    [Google Scholar]
  9. Colberg-Poley, A. M., Huang, L., Soltero, V. E., Iskenderian, A., Schumacher, R.-F. & Anders, D. G. ( 1998; ). The acidic domain of pUL37x1 and gpUL37 plays a key role in transactivation of HCMV DNA replication gene promoter constructions. Virology 246, 400–408.[CrossRef]
    [Google Scholar]
  10. Colberg-Poley, A. M., Patel, M. B., Erezo, D. P. P. & Slater, J. E. ( 2000; ). Human cytomegalovirus immediate early regulatory proteins traffic through the secretory apparatus and to mitochondria. J Gen Virol 81, 1779–1789.
    [Google Scholar]
  11. Colgan, D. F. & Manley, J. L. ( 1997; ). Mechanism and regulation of mRNA polyadenylation. Genes & Dev 11, 2755–2766.[CrossRef]
    [Google Scholar]
  12. Cote, J. & Chabot, B. ( 1997; ). Natural base-pairing interactions between 5′ splice site and branch site sequences affect mammalian 5′ splice site selection. RNA 3, 1248–1261.
    [Google Scholar]
  13. Deguillien, M., Huang, S.-C., Morinière, M., Dreumont, N., Benz, E. J., Jr & Baklouti, F. ( 2001; ). Multiple cis elements regulate an alternative splicing event in 4.1R pre-mRNA during erythroid differentiation. Blood 98, 3809–3816.[CrossRef]
    [Google Scholar]
  14. Fu, X. D. ( 1995; ). The superfamily of arginine/serine-rich splicing factors. RNA 1, 663–680.
    [Google Scholar]
  15. Goldmacher, V. S., Bartle, L. M., Skaletskaya, A. & 10 other authors ( 1999; ). A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to bcl-2. Proc Natl Acad Sci U S A 96, 12536–12541.[CrossRef]
    [Google Scholar]
  16. Hastings, M. L. & Krainer, A. R. ( 2001; ). Pre-mRNA splicing in the new millennium. Curr Opin Cell Biol 13, 302–309.[CrossRef]
    [Google Scholar]
  17. Hayajneh, W. A., Colberg-Poley, A. M., Skaletskaya, A., Bartle, L. M., Lesperance, M. M., Contopoulos-Ioannidis, D. G., Kedersha, N. L. & Goldmacher, V. S. ( 2001a; ). The sequence and antiapoptotic functional domains of the human cytomegalovirus UL37 exon 1 immediate early protein are conserved in multiple primary strains. Virology 279, 233–240.[CrossRef]
    [Google Scholar]
  18. Hayajneh, W. A., Contopoulos-Ioannidis, D. G., Lesperance, M. M., Venegas, A. & Colberg-Poley, A. M. ( 2001b; ). The carboxyl terminus of the human cytomegalovirus UL37 immediate-early glycoprotein is conserved in primary strains and is important for transactivation. J Gen Virol 82, 1569–1579.
    [Google Scholar]
  19. Kouzarides, T., Bankier, A. T., Satchwell, S. C., Preddy, E. & Barrell, B. G. ( 1988; ). An immediate early gene of human cytomegalovirus encodes a potential membrane glycoprotein. Virology 165, 151–164.[CrossRef]
    [Google Scholar]
  20. Krämer, A. ( 1996; ). The structure and function of proteins involved in mammalian pre-mRNA splicing. Annu Rev Biochem 65, 367–409.[CrossRef]
    [Google Scholar]
  21. Lee, M., Xiao, J., Haghjoo, E., Zhan, X., Abenes, G., Tuong, T., Dunn, W. & Liu, F. ( 2000; ). Murine cytomegalovirus containing a mutation at open reading frame M37 is severely attenuated in growth and virulence in vivo. J Virol 74, 11099–11107.[CrossRef]
    [Google Scholar]
  22. Le Hir, H., Izaurralde, E., Maquat, L. E. & Moore, M. J. ( 2000; ). The spliceosome deposits multiple proteins 20–24 nucleotides upstream of mRNA exon–exon junctions. EMBO J 19, 6860–6869.[CrossRef]
    [Google Scholar]
  23. Lin, C.-H. & Patton, J. G. ( 1995; ). Regulation of alternative 3′ splice site selection by constitutive splicing factors. RNA 1, 234–245.
    [Google Scholar]
  24. McCullough, A. J. & Schuler, M. A. ( 1997; ). Intronic and exonic sequences modulate 5′ splice site selection in plant nuclei. Nucleic Acids Res 25, 1071–1077.[CrossRef]
    [Google Scholar]
  25. MacDonald, C., Wilusz, J. & Shenk, T. ( 1994; ). The 64 kilodalton subunit of CstF polyadenylation factor binds to pre-mRNAs downstream of the cleavage site and influences cleavage site location. Mol Cell Biol 14, 6647–6654.
    [Google Scholar]
  26. Reed, R. ( 1989; ). The organization of the 3′ splice-site sequences in mammalian introns. Genes & Dev 3, 2113–2123.[CrossRef]
    [Google Scholar]
  27. Reed, R. & Maniatis, T. ( 1985; ). Intron sequences involved in lariat formation during pre-mRNA splicing. Cell 41, 95–105.[CrossRef]
    [Google Scholar]
  28. Ruskin, B. & Green, M. R. ( 1985; ). Role of the 3′ splice site consensus sequence in mammalian pre-mRNA splicing. Nature 317, 732–734.[CrossRef]
    [Google Scholar]
  29. Singh, R., Valcárcel, J. & Green, M. R. ( 1995; ). Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins. Science 268, 1173–1176.[CrossRef]
    [Google Scholar]
  30. Skaletskaya, A., Bartle, L. M., Chittenden, T., McCormick, A. L., Mocarski, E. S. & Goldmacher, V. S. ( 2001; ). A cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation. Proc Natl Acad Sci U S A 98, 7829–7834.[CrossRef]
    [Google Scholar]
  31. Smith, J. A. & Pari, G. S. ( 1995; ). Expression of human cytomegalovirus UL36 and UL37 genes is required for viral DNA replication. J Virol 69, 1925–1931.
    [Google Scholar]
  32. Smith, C. W. J., Porro, E. B., Patton, J. G. & Nadal-Ginard, B. ( 1989; ). Scanning from an independently specified branch point defines the 3′ splice site of mammalian introns. Nature 342, 243–247.[CrossRef]
    [Google Scholar]
  33. Tarn, W.-Y. & Steitz, J. A. ( 1995; ). Modulation of 5′ splice site choice in pre-messenger RNA by two distinct steps. Proc Natl Acad Sci U S A 92, 2504–2508.[CrossRef]
    [Google Scholar]
  34. Tenney, D. J. & Colberg-Poley, A. M. ( 1990; ). RNA analysis and isolation of cDNAs derived from the human cytomegalovirus immediate early region at 0·24 map units. Intervirology 31, 203–214.
    [Google Scholar]
  35. Tenney, D. J. & Colberg-Poley, A. M. ( 1991a; ). Expression of the human cytomegalovirus UL36-38 immediate early region during permissive infection. Virology 182, 199–210.[CrossRef]
    [Google Scholar]
  36. Tenney, D. J. & Colberg-Poley, A. M. ( 1991b; ). Human cytomegalovirus UL36-38 and US3 immediate early loci RNA: temporal expression of nuclear, cytoplasmic, and polysome associated transcripts during infection. J Virol 65, 6724–6734.
    [Google Scholar]
  37. Tenney, D. J., Santomenna, L. D., Goudie, K. B. & Colberg-Poley, A. M. ( 1993; ). The human cytomegalovirus US3 immediate early protein lacking the putative transmembrane domain regulates gene expression. Nucleic Acids Res 21, 2931–2937.[CrossRef]
    [Google Scholar]
  38. Umen, J. G. & Guthrie, C. ( 1995; ). A novel role for a U5 snRNP protein in 3′ splice site selection. Genes & Dev 9, 855–868.[CrossRef]
    [Google Scholar]
  39. Wahle, E. & Keller, W. ( 1996; ). The biochemistry of polyadenylation. Trends Biochem Sci 21, 247–250.[CrossRef]
    [Google Scholar]
  40. Wassarman, D. A. & Steitz, J. A. ( 1992; ). Interactions of small nuclear RNAs with precursor messenger RNA during in vitro splicing. Science 257, 1918–1925.[CrossRef]
    [Google Scholar]
  41. Wu, S. & Green, M. R. ( 1997; ). Identification of a human protein that recognizes the 3′ splice site during the second step of pre-mRNA splicing. EMBO J 16, 4421–4432.[CrossRef]
    [Google Scholar]
  42. Wu, S., Romfo, C. M., Nilsen, T. W. & Green, M. R. ( 1999; ). Functional recognition of the 3′ splice site AG by the splicing factor U2AF35. Nature 402, 832–835.[CrossRef]
    [Google Scholar]
  43. Zhu, H., Cong, J.-P., Mamtora, G., Gingeras, T. & Shenk, T. ( 1998; ). Cellular gene expression altered by human cytomegalovirus: global monitoring with oligonucleotide arrays. Proc Natl Acad Sci U S A 95, 14470–14475.[CrossRef]
    [Google Scholar]
  44. Zhuang, Y. & Weiner, A. M. ( 1990; ). The conserved dinucleotide AG of the 3′ splice site may be recognized twice during in vitro splicing of mammalian mRNA precursors. Gene 90, 263–269.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.18700-0
Loading
/content/journal/jgv/10.1099/vir.0.18700-0
Loading

Data & Media loading...

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error